Sustainable carbon dots from Borreria hispida: enhanced colorimetric sensing of Fe3+ ions and biological applications in live cell imaging

This study presents the synthesis of advanced nanomaterials derived from the hedge-grown herbal plant, Borreria hispida, and explores their environmental and biological applications. Using a one-step hydrothermal synthesis method, carbon dots derived from Borreria hispida (BHCD) were fabricated and thoroughly characterized through XRD, TEM, FTIR, CHNS, UV-visible, and PL spectroscopy analyses. Under UV illumination, these plant-based carbon dots demonstrated exceptional water solubility, notable photo stability, and a high quantum yield of 40.8%. The average particle size of BHCD was absorbed around 0.5 to 3.5 nm, contributing to superior selectivity and sensitivity in detecting Fe3+ ions, with a limit of detection of 1.2 × 10−6 M. Investigation into the sensing mechanism revealed a binding model wherein two carbon atom molecules bind to one Fe3+ atom in a 2 : 1 ratio for BHCDs and Fe3+ interactions. Additionally, the effectiveness of the developed fluorescent probe for Fe3+ detection was validated using real water samples from ponds and lakes, highlighting its potential for environmental monitoring applications. Furthermore, the biological effects of BHCD were evaluated through cytotoxic assays, demonstrating significant inhibitory effects on MCF7 breast cancer cell lines, with a maximum cell viability of 60%. This research underscores the multifaceted potential of BHCD in environmental monitoring and biomedical applications.


Introduction
Iron, an essential element for life and classied as a transition metal, poses a signicant threat to human health due to the increasing presence of Fe 3+ ions in water resources.Elevated levels of Fe 3+ in the human body have been linked to various health issues, 1,2 including organ malfunctions such as those in the heart, pancreas, and liver.Multiple sources contribute to the rise of iron in drinking water, including the dissolution of ironstones, contamination from industrial wastewater, corrosion of water pipes, and the use of construction materials in drinking water resources. 3,4High concentrations of iron lead to water turbidity, while low concentrations promote bacterial growth, resulting in clogged water supply pipelines and unpleasant odours.Furthermore, a heightened iron concentration in water serves as an indicator for other heavy metals. 5,6he prevalence of iron in the environment and water resources surpasses that of other heavy metal ions like lead and mercury.[9] To achieve sustainable progress, it is crucial to recognize and eliminate harmful pollutants.1][12] Promising technologies for detecting and eliminating toxic contaminants utilize uorescence sensing and photo-degradation-based methods.7][18] Through successful endeavours, they have synthesized quasi-spherical nano-sized CDs (<10 nm) using both top-down and bottom-up approaches.0][21] In nanomaterial synthesis, topdown methodologies involve fragmenting carbon matter into carbon nanoparticles using diverse techniques such as arc discharge, laser ablation, electrochemistry, and wet oxidation.][22] Researchers achieve this conversion using hydrothermal,

RSC Advances
4][25] Among the bottom-up approaches, pyrolysis is widely employed.7][28] These CDs fall under sustainable nanotechnology, given the prevalent use of various plants, fruits, or bio-waste as carbon precursors. 29These CDs nd vast and impactful applications across diverse elds such as bioimaging, cancer therapy, drug delivery, optoelectronic devices (including solar cells and light-emitting devices), catalysis, supercapacitors, agriculture, and optical sensors designed for detecting pollutants and heavy metals. 30o bridge the gap, we propose exploring the synthesis of carbon dots derived from hedge-grown plants.We will investigate these carbon dots, previously reported from edible plants like bananas and well-known herbal plants like Tulsi, for their environmental and biological applications, as work has yet to be reported on hedge-grown plants, particularly this plant (Borreria hispida).This study promises to contribute signicantly to the eld by expanding the path of carbon dots sources and unlocking new possibilities for their utilization in diverse technological and biomedical applications.By exploring this unreported approach in Borreria hispida, we aim to ll a critical void in the existing body of knowledge and pave the way for future advancements in carbon dot research.The novelty of this material lies not only in its botanical origin but also in its unexplored potential for specic applications such as metal sensing and bioimaging.

Characterization techniques
Various analytical methods were applied to the acquired BHCD.These encompassed Fourier Transform Infrared Spectroscopy (HITACHI), UV-visible absorbance spectroscopy (Jasco V-670) to establish the excitation range of the sample, and the examination of uorescence properties using Fluorescence Spectroscopy (HITACHI F7000).Elemental analysis, determining the percentages of carbon, hydrogen, nitrogen, and sulfur in carbon dots, was conducted using a PerkinElmer −2400 CHNS/O series.The material's structure was elucidated through XRD analysis using a Bruker D8 Advance, while SEM analysis was carried out utilizing a Carl Zeiss EVO/18 Research microscope.High-Resolution Transmission Electron Microscopy (HR-TEM) was conducted using the FEI-Tecnai G2 20 Twin instrument, and Xray Photoelectron Spectroscopy (XPS) was performed with the ULVAC-PHI Versa Probe 4. The synergy H1 microplate reader (Biotek) conducted the MTT assay, and uorescence images were captured using the RTC-7 CON inverted uorescence microscope.

Sample preparation for sensing
We used distilled water to prepare a stock solution of BHCD (1 × 10 −2 mol L −1 ).Fe 3+ was generated by dissolving FeCl 3 in water to achieve a concentration of 0.1 mol L −1 .Additionally, various anion stock solutions (Se 2+ , Bi 2+ , Li 2+ , Ni 2+ , Fe 3+ , Na 2+ , Cr 2+ , Ba 2+ , Ca 2+ , Cu 2+ ) were prepared using distilled water at a concentration of 0.1 mol L −1 .These solutions were diluted to attain a uniform 1 × 10 −2 mol L −1 concentration.All subsequent optical spectral characterization studies utilized these precisely calibrated stock solutions, ensuring the accuracy and reliability of our experimental setup.This preparation establishes a solid foundation for further analytical investigations.

Plant selection and collection
We selected Borreria hispida among the hedge-grown herbal plants for our work due to its remarkable therapeutic properties and reported pharmacological activities.The methanolic extract of Borreria hispida seeds demonstrated anticancer activity, 31 while the leaves exhibited anti-inammatory activity. 32Consequently, we decided to explore the novel application of Borreria hispida-derived carbon dots for metal sensing and cell imaging.In August, we collected the plant in Village C.N. Pattadai, Vellore, Tamil Nadu, India.The plant, fully matured and grown in loam soil, was shade-dried for two weeks, powdered using an electric grinder, and stored for further use.

Synthesis of carbon dots (BHCD)
To synthesize carbon dots using the one-pot hydrothermal method with Borreria hispida, dissolve 5 g of powdered leaves of Borreria hispida in 125 mL of deionized water.Stir the mixture for 10 minutes and subject it to 10 minutes of sonication.Transfer the solution into a Teon-lined autoclave and place it in a muffle furnace at 180 °C for 4 hours.Aerward, lter the solution using Whatman lter paper.Take the ltrate to separate large molecules through the dialysis method.Pack the solution into a membrane bag and allow it to contact Milli-Q water for purication over 14 hours.Finally, refrigerate the puried solution at 4 °C for future use.Scheme 1 explains this one-step synthesis using an herbal plant for producing sustainable carbon dots.

Structural characterization
The utilization of Borreria hispida, identiable by its white owers, holds substantial promise as a natural source for producing carbon quantum dots (CQDs) owing to its abundant carbon, oxygen, and nitrogen elements across the entire plant structure.We employed a one-step hydrothermal synthesis method, yielding a CQD solution displaying a faint brown colour, indicative of the successful carbonization of the Borreria hispid plant material.Notably, the CQD solution showcased a measured pH of approximately 6, suggesting the presence of surface functional groups such as -OH and -COOH.
The interpreted peaks from the Fourier Transform Infrared (FT-IR) spectra, illustrated in Fig. 1(a), are compelling evidence conrming the diverse chemical functionalities within the CQDs.Specically, a medium peak observed around 3343 cm −1 implies the stretching of O-H and N-H bonds, indicating the presence of aliphatic primary amines.Additionally, the 1636 cm −1 and 1403 cm −1 peaks correspond to the C]O stretching and COO-stretching vibration.
To obtain X-ray diffraction (XRD) patterns for Thin-Layer Chromatography Liquid Crystal Displays (BHCDs), we applied a solution of BHCDs onto a pristine glass slide, le it to desiccate overnight at 50 °C, resulting in the formation of a thin lm, as depicted in Fig. 1(b).We determined the interplanar distance (d-spacing) values for BHCDs using Bragg's equation, involving parameters such as the value of theta, which is 23.85, and the plane value of 002, consistent with that previously reported for CDs (position of the plane), n (a positive integerin this case, 1), and l (wavelength of the incident X-rays, where l = 1.54 Å).Furthermore, we computed interatomic distances (a), with 'd' representing the interplanar distance and h, k, and l denoting the Miller indices.
In Fig. 3, we employed X-ray photoelectron spectroscopy (XPS) to analyse the synthesized carbon quantum dots (CQDs) for surface functional groups, chemical composition, and elemental states.The XPS full survey spectrum of CQDs showed distinct peaks at 529.

Optical properties
The UV-vis spectra revealed two distinct bands, each conveying signicant transitions.At 210 nm, a distinct band indicated the p to p* transition, characterized by higher energy levels as shown in Fig. 6.Conversely, the band at 288 nm corresponded to the n to p* transition, similarly denoting higher energy involvement.The different concentrations of BHCD in different regions as shown in Fig. 5. Intriguingly, the uorescence spectra exhibited an emission peak at 475 nm.Notably, the naked eye immediately and noticeably perceived a change in colour upon observing the BHCDs compound, shiing from a light brown shade to a vibrant yellow shade.
Fluorescence spectroscopy analysis of the synthesized BHCDs is conducted under various wavelengths, as depicted in Fig. 7. Exciting CDs with shorter wavelengths increase their energy, enabling electrons to transition to higher vibrational states this reduction in emission energy results in an enhancement of Stokes shis and a decrease in uorescence intensity.The peak position shis towards longer wavelengths as the excitation wavelength ranges from 240 to 480 nm.

Metal ion sensing
Selectivity studies.We utilized uorescence spectroscopy for selectivity studies to evaluate the metal ion sensing capabilities of BHCDs.Aqueous solutions of various metal ions (Se 2+ , Bi 2+ , Li 2+ , Ni 2+ , Fe 3+ , Na 2+ , Cr 2+ , Ba 2+ , Ca 2+ , Cu 2+ ) were prepared using their respective chemical salts.The objective was to investigate the selectivity of BHCDs concerning these metal ions.We incubated 0.2 mL of each metal ion for these experiments with a 1.8 mL solution of diluted BHCDs.This incubation lasted for 15 minutes, resulting in a nal volume of 2 mL for each solution and a concentration of 200 mM for every metal ion.During this examination, Fig. 8 illustrates the absorbance, and the response of Fe 3+ ions was particularly intriguing.Notably, the presence of Fe 3+ induced a red shi in the uorescence spectra, suggesting a distinctive response from BHCDs in the presence of this specic metal ion.However, observations indicated that other metals did not induce signicant changes in the absorbance spectra, implying a lack of substantial interaction or response from BHCDs in their presence.Fig. 9(i) and (ii) show noticeable colour changes in visible and UV light images upon detecting Fe 3+ metal using BHCD and different concentrations.The observed changes demonstrate BHCD's sensitivity to Fe 3+ , with clear visible and UV spectra alterations.This visual representation emphasizes the effectiveness of BHCD as a Fe 3+ detection sensor.
Sensitivity studies.To conrm the interaction between Fe 3+ ions and BHCDs, we investigated thoroughly to identify potential complex formation between the metal ions and the compound.Our focus cantered on examining the absorption spectra of BHCDs following the incremental addition of Fe 3+ ions.The absorption band at 204 nm gradually decreased, while a new band emerged at 309 nm as shown in Fig. 10.This shi in the spectra strongly suggests a dynamic interaction between Fe 3+ ions and BHCDs, with the diminishing intensity at 204 nm indicating a decline in the initial absorption band coinciding with the addition of Fe 3+ ions.
Simultaneously, a distinct absorption band at 309 nm hints at forming a new entity resulting from the interaction between Fe 3+ ions and BHCDs.This spectral evolution strongly suggests the potential formation of a compound due to the interaction between the metal ions and BHCDs, shedding light on the intricate nature of their interplay.
Binding studies.To investigate the binding affinity of Fe 3+ ions with BHCDs, we conducted a comprehensive analysis using Job's plot, meticulously tracking absorbance changes across varying Fe 3+ ion concentrations.
Furthermore, we determined the detection limit (LOD) and quantication limit (LOQ) using the standard formula Ks/s, with K values of 3 for LOD and 10 for LOQ, as shown in Fig. 12 By utilizing Job's plot, we successfully ascertained the stoichiometric ratio of BHCDs to Fe 3+ ions as 2 : 1 as shown in Fig. 13.Moreover, we deduced the binding constant by assessing the ratio between the intercept and slope derived from observable colour changes upon the addition of Fe 3+ ions to BHCDs under visible light conditions.The construction of the Bansi-Hildebrand plot, illustrated in Fig. 11 using the standard BH formula, claries this binding constant determination   process.The quantication of the binding efficiency of Fe 3+ ions with BHCDs was calculated as 4.77 × 10 2 M −1 , underscoring the robust binding affinity and selectivity of BHCDs, specically for Fe 3+ ions.This rigorous analysis illuminates the profound interaction and binding capabilities of BHCDs towards Fe 3+ ions.pH studies.Nanoparticles signicantly inuence the stability and sensing capabilities, with the pH of the medium playing a crucial role.To enhance the sensing potential of BHCDs for Fe 3+ , we conducted experiments across a pH range of 1 to 12, controlling the pH using NaOH (0.1 M) for alkaline conditions and HCl (0.1 M) for acidic conditions.We recorded absorption spectra at room temperature, and Fig. 14 displays the absorbance vs. pH graph, demonstrating that BHCDs effectively recognize Fe 3+ over a broad pH spectrum.We conducted further investigations to determine the binding stoichiometry of the BHCDs-iron complex.
Interference studies.We aimed to explore the interference potential of various anions on the accurate detection of iron metal by monitoring alterations in the absorption spectrum of the BHCD-Fe 3+ solution.To assess this, we introduced 10 equivalents of diverse competing metals, encompassing Se 2+ , Bi 2+ , Li 2+ , Ni 2+ , Na 2+ , Cr 2+ , Ba 2+ , Ca 2+ , and Cu 2+ ions.Interestingly as shown in Fig. 15, our observations revealed no discernible shis in spectral changes or deviations in absorption maxima within the BHCD-Fe 3+ solution, particularly at 345 nm.This recent project signies the development of a sensing probe, displaying a signicant leap forward compared to previously documented chemical-based probes.This exploration highlights the robustness and specicity of the BHCD-Fe 3+ system as a potential sensing tool, offering enhanced accuracy in hypochlorite detection and setting a new benchmark in sensing technology.
Mechanism of Fe 3+ sensing by BHCD.FT-IR spectroscopy revealed the presence of functional groups in the carbon dots.
The C]O and COO-groups were conrmed at peaks around 1636 cm −1 and 1403 cm −1 , respectively, while the O-H and N-H groups were identied at a peak around 3343 cm −1 , suggesting the existence of secondary amines.In XPS analysis, the aldehyde (C-OH) group was identied at a binding energy of 286.2 eV in the C 1s spectrum.The ketone (C]O) and aldehyde (C-OH) groups were also observed at binding energies of 531.2 eV and 523.3 eV, respectively.The presence of the secondary amine (N-H) group was conrmed at a binding energy of 401.2 eV.Carbon, nitrogen, and oxygen were detected in the carbon dots, indicating the presence of functional groups such as OH, carbonyl (aldehyde, ketone), and amine.All these functional groups contain lone pairs that can donate electrons to the metal's empty d orbitals, forming coordinate bonds.This    interaction results in changes in absorbance, providing insight into the mechanism through which carbon dots detect Fe 3+ .
Real-time water analysis.We evaluated the practical utility of BHCDs in detecting specic metal ions using tap water samples collected from our laboratory and pond and lake water obtained from Jambukulam, Vellore, spiked with a known concentration (50 mM) of Fe 3+ ions.We ltered the water samples using 20 nm lter paper to remove impurities.Subsequently, we introduced varying concentrations of Fe 3+ , ranging from 0 to 20 mM, into the ltered samples.We measured the emission intensity at 475 nm during excitation to determine the Fe 3+ concentration as shown in Fig. S2.† A standard UV-vis spectrum was a reference to validate the Fe 3+ concentration in actual water samples.By comparing the emission intensities obtained from the water samples with the calibration curve, we estimated the corresponding Fe 3+ concentrations.The comprehensive analysis of diverse water samples illustrated the accurate detection of the target compound.Importantly, we achieved recovery rates ranging from 98.6% to 104.3% using the BHCDs probe for Fe 3+ detection, robustly conrming the efficiency of the BHCDs probe in the precise and quantitative detection of Fe 3+ in realworld water samples.

Bio imaging
The cytotoxic effects of Borreria hispida derived carbon dots (BHCD) were studied using MCF7 (Michigan Cancer Foundation-7) cell lines, known to cause breast cancer in humans.For this study, BHCD concentrations ranging from 62.5 to 500 mg mL −1 were examined via the MTT assay to evaluate their impact on cell viability.Cytotoxicity tests were conducted on MCF-7 cells treated with BHCD at 62.5, 125, 250, and 500 mg mL −1 concentrations, as depicted in Fig. 16.At the maximum concentration of BHCD (500 mg mL −1 ), cell viability was observed to be 60%, as illustrated in Fig. 17.
Therefore, we suggest that BHCD exhibits a good level of cytotoxicity.The 60% cell viability result indicates that these carbon dots possess the potential for cytotoxicity against MCF7 cell lines.
Additionally, Fig. 18c and d display IC50 and maximum concentration of BHCD-treated MCF7 cells, showing a color change from dark green to light green as shown in Fig. 18.The IC50 concentration results in a partial color change, while the maximum concentration induces a complete color change compared to the control.The loss in membrane integrity and apoptotic induction, indicated by light green fragmented cells, is more pronounced in the maximum concentration group compared to the other treated cell lines exhibiting color changes in increasing order.The investigation conrms that BHCD-induced apoptosis is linked to ROS formation.From this assay, we conclude that increasing concentration correlates with an increasing color change, reecting the cytotoxicity of BHCD against MCF7 cell lines.

Conclusion
The synthesis of Borreria hispida carbon dots (BHCDs) from plant leaves has yielded remarkable nanomaterials with exceptional physicochemical properties.With an average particle size of merely 3.33 nm, high water solubility, remarkable photostability, and a substantial quantum yield of 40.8%, they   demonstrate immense potential as a colorimetric probe for Fe 3+ detection.The binding model, revealing a 2 : 1 ratio of BHCDs through a jobs plot, further accentuates their high selectivity and sensitivity, achieving an impressively low detection limit for Fe 3+ ions.Validation using real water samples from natural environments such as ponds and lakes conrms the effectiveness of these BHCDs for practical environmental monitoring applications, positioning them as promising tools for sensitive and selective Fe 3+ detection in real-world scenarios.The anticancer activity of BHCD against MCF7 breast cancer cell lines and the resulting 60% cell viability indicate a signicant potential in biological applications.
Scheme 1 Synthetic route of Borreria hispida based carbon dots through hydrothermal reaction.

Fig. 8
Fig. 8 Selectivity studies of BHCD with various metal cations.

Fig. 14
Fig. 14 pH studies of BHCD with Fe 3+ in different pH levels.

Fig. 13
Fig. 13 Jobs plot for BHCD in different mole fraction of Fe 3+ .